Differentiated human airway organoids to assess infectivity of emerging influenza virus

Zhou, Jie; Li, Cun; Sachs, Norman; Chiu, Man Chun; Wong, Bosco Ho-Yin; Chu, Hin; Poon, Vincent Kwok-Man; Wang, Dong; Zhao, Xiaoyu; Wen, Lei; Song, Wenjun; Yuan, Shuofeng; Wong, Kenneth Kak-Yuen; Chan, Jasper Fuk-Woo; To, Kelvin Kai-Wang; Chen, Honglin; Clevers, Hans; Yuen, Kwok-Yung

Novel reassortant avian influenza H7N9 virus and pandemic 2009 H1N1 (H1N1pdm) virus cause human infections, while avian H7N2 and swine H1N1 virus mainly infect birds and pigs, respectively. There is no robust in vitro model for assessing the infectivity of emerging viruses in humans. Based on a recently established method, we generated long-term expanding 3D human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. We report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. In addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. We also established improved 2D monolayer culture conditions for the differentiated airway organoids. To demonstrate the ability of differentiated airway organoids to identify human-infective virus, 3D and 2D differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, H7N9/Ah versus H7N2 and H1N1pdm versus an H1N1 strain isolated from swine (H1N1sw). The humaninfective H7N9/Ah virus replicated more robustly than the poorly human-infective H7N2 virus; the highly human-infective H1N1pdm virus replicated to a higher titer than the counterpart H1N1sw. Collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. These differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human. airway organoid | proximal differentiation | influenza virus | infectivity I nfluenza A viruses (IAVs) can infect a diversity of avian and mammalian species, including humans, and have the remarkable capacity to evolve and adapt to new hosts (1) . The segmented RNA genomes of IAVs and the low fidelity of RNA polymerase allow antigenic shift and drift, which drive the viral evolution. Thus, novel viruses from birds and pigs cross the species barrier and infect humans, leading to sporadic infections, epidemics, and even pandemics (1, 2) . Despite the tremendous progress made in virology and epidemiology, which subtype or strain of IAV will cause the next outbreak remains unpredictable. A novel reassortant H7N9 influenza virus from poultry has led to recurrent outbreaks of human infection in China since 2013 (2, 3) . More than 1,500 laboratory-confirmed cases of H7N9 human infections were reported by October 2017, with a case-fatality rate higher than 35% (4). In 2009, the first influenza pandemic of the 21st century was caused by a novel pandemic H1N1 (H1N1pdm), which originated via multiple reassortments of "classical" swine H1N1 virus with human H3N2 virus, avian virus, and avian-like swine virus (5). While swine viruses infect humans only sporadically, this novel strain of swine-derived H1N1pdm virus has infected a large proportion of the human population, establishing sustained human-to-human transmission and circulating globally as a seasonal virus strain since then.